As used herein, the terra “blood products” includes whole blood and cellular components derived from blood, including erythrocytes (red blood cells) and platelets.
There are more than thirty blood group or type) systems, one of the most important of which is the ABO system. This system is based on the presence or absence of antigens A and/or B. These antigens are found on the surface of erythrocytes and platelets as well as on the surface of endothelial and most epithelial cells. The major blood product used for transfusion is erythrocytes, which are red blood cells containing hemoglobin, the principal function of which is the transport of oxygen. Blood of group A contains antigen A on its erythrocytes. Similarly, blood of group B contains antigen B on its erythrocytes. Blood of group AB contains both antigens, and blood of group O contains neither antigen.
The blood group structures are glycoproteins or glycolipids and considerable work has been done to identify the specific structures making up the A and B determinants or antigens. The ABH blood group specificity is determined by the nature and linkage of monosaccharides at the ends of the carbohydrate chains. The carbohydrate chains are attached to a peptide (glycoprotein) or lipid (glycosphingolipid) backbone, which are attached to the cell membrane of the cells. The immunodominant monosaccharide determining type A specificity is a terminal α1-3 linked N-acetylgalactosamine (GalNAc), while the corresponding monosaccharide of B type specificity is an α1-3 linked galactose (Gal). Type O cells lack either of these monosaccharides at the termini of oligosaccharide chains, which instead are terminated with α1-2 linked fucose (Fuc) residues.
A great diversity of blood group ABH carbohydrate structures are found due to structural variations in the oligosaccharide chains that carry ABH immunodominant saccharides. Table 1 lists structures reported in man and those that have been found on human red cells or in blood extracts. For a review, see, Clausen & Hakomori, Vox Sang 56(1): 1-20, 1989). Red cells contain ABH antigens on N-linked glycoproteins and glycosphingolipids, while it is generally believed that O-linked glycans on erythrocytes glycoproteins, mainly glycophorins, are terminated by sialic acid and not with ABH antigens. Type 1 chain glycosphingolipids are not endogenous products of red cells, but rather adsorbed from plasma.
TABLE IHisto-Blood Group ABH Immunoreactive Determinants of Human Cells1Type ofFound onStructureNameHapten StructureGlycoconjugateRBCNoA type 1, ALedGlycolipid N-linked O-linkedGlycolipid1 A type 1, ALebGlycolipid N-linked O-linkedGlycolipid2 A type 2, AGlycolipid N-linked O-linkedGlycolipid N-linked3 A type 2, ALevGlycolipid N-linked O-linkedGlycolipid?4 A type 3, O-linkedO-linked5 A type 3, RepetitiveGlycolipidGlycolipid6 A type 4, GloboGlycolipidGlycolipid?7 A type 4, GanglioGlycolipid8 B type 1, BLedGlycolipid N-linked O-linkedGlycolipid9 B type 1, BLebGlycolipid N-linked O-linkedGlycolipid10 B type 2, BGlycolipid N-linked O-linkedGlycolipid N-linked11 B type 2, BLeyGlycolipid N-linked O-linkedGlycolipid?12 B type 3, O-linkedO-linked13 B type 4, GloboGlycolipid?Glycolipid?14 B type 4, GanglioGlycolipid?15 H type 1, LedGlycolipid N-linked O-linkedGlycolipid16 H type 1, LebGlycolipid N-linked O-linkedGlycolipid17 H type 2, HGlycolipid N-linked O-linkedGlycolipid N-linked18 H type 2, LeyGlycolipid N-linked O-linkedGlycolipid?19 H type 3, O-linkedO-linked20 H type 3, H-AGlycolipidGlycolipid (A RBC)21 H type 4, GloboGlycolipidGlycolipid22 H type 4, GanglioGlycolipid23 Thomsen-FriedenrichGalβ1-3GalNAcα1-O-Ser/ThrO-linkedO-linked24Tf, T(+SA) Gal-A, T cross-react.GlycolipidGlycolipid (A RBC)25 Tn, A cross-react.GalNAcα1-O-Ser/ThrO-linkedO-linked26(+SA)1Adapted from Clausen and Hakomori, Vox Sang 56(1): 1-20, 1989.Designations: “?” indicates potential gycolipid structures which have not been reported to date.
Blood group A and B exist in several subtypes. Blood group A subtypes are the most frequent, and there are three recognized major sub-types of blood type A. These sub-types are known as A1, A intermediate (Aint) and A2. There are both quantitative and qualitative differences that distinguish these three sub-types. Quantitatively, A1 erythrocytes have more antigenic A sites, i.e., terminal N-acetylgalactosamine residues, than Aint erythrocytes which in turn have more antigenic A sites than A2 erythrocytes. Qualitatively, A1 erythrocytes have a dual repeated A structure on a subset of glycosphingolipids, while A2 cells have an H structure on an internal A structure on a similar subset of glycolipids (Clausen et al., Proc. Natl. Acad. Sci. USA 82(4): 1199-203, 1985, Clausen et al., J. Biol. Chem. 261(3): 1380-7, 1986). These differences between A1 and weak A subtypes are thought to relate to differences in the kinetic properties of blood group A isoenzyme variants responsible for the formation of A antigens (Clausen et al., J. Biol. Chem. 261(3): 1388-92, 1986). The differences of group B subtypes are believed to be solely of quantitative nature.
Blood of group A contains antibodies to antigen B. Conversely, blood of group B contains antibodies to antigen A. Blood of group AB has neither antibody, and blood group O has both. Antibodies to these and other carbohydrate defined blood group antigens are believed to be elicited by continuous exposure to microbial organism carrying related carbohydrate structures. An individual whose blood contains either (or both) of the anti-A or anti-B antibodies cannot receive a transfusion of blood containing the corresponding incompatible antigen(s). If an individual receives a transfusion of blood of an incompatible group, the blood transfusion recipient's antibodies coat the red blood cells of the transfused incompatible group and cause the transfused red blood cells to agglutinate, or stick together. Transfusion reactions and/or hemolysis the destruction of red blood cells) may result therefrom.
In order to avoid red blood cell agglutination, transfusion reactions, and hemolysis, transfusion blood type is cross-matched against the blood type of the transfusion recipient. For example, a blood type A recipient can be safely transfused with type A blood, which contains compatible antigens. Because type O blood contains no A or B antigens, it can be transfused into any recipient with any blood type, i.e., recipients with blood types A, B, AB or O. Thus, type O blood is considered “universal”, and may be used for all transfusions. Hence, it is desirable for blood banks to maintain large quantities of type O blood. However, there is a paucity of blood type O donors. Therefore, it is desirable and useful to remove the immunodominant A and B antigens on types A, B and AB blood in order to maintain large quantities of universal blood products.
In an attempt to increase the supply of type O blood, methods have been developed for converting certain type A, B and AB blood to type O blood. Conversion of B cells to type O cells has been accomplished in the past. However, conversion of the more abundant A cells has only been achieved with the less abundant weak A subgroup cells. The major obstacle for development and utilization of enzyme converted universal O cells has, in the past, been the failure to enzymatically convert the strong A1 cells. This obstacle has remained. As will be explained below in detail the enzymes and methods used in the prior art are inefficient, impractical, and/or too costly to be used in a commercial process to supply universal type O cells.
Conversion of B Cells:
Enzymatic conversion of type B blood using purified or recombinant coffee bean (Coffey canephora) α-galactosidase has been achieved using 100-200 U/ml (U.S. Pat. No. 4,427,777; Zhu et al., Arch Biochem Biophys 1996; 327(2): 324-9; Kruskall et al., Transfusion 2000; 40(11): 1290-8). The specific activity of coffee bean α-galactosidase was reported to be 32 U/mg using p-nitrophenyl α-D-Gal with one unit (U) defined as one μmole substrate hydrolyzed per minute (Zhu et al., Arch Biochem Biophys 1996; 327(2): 324-9). Enzymatic conversions were done at pH 5.5 with approximately 6 mg/ml enzyme at 80-90% hematocrit, and the resulting converted O cells functioned normally in transfusion experiments and no significant adverse clinical parameters were observed (Kruskall et al., Transfusion 2000; 40(11): 1290-8). This data along with earlier publications, clearly demonstrate that enzymatic conversion of red blood cells is feasible and that such enzyme group B converted O (B ECO) cells can function as well as matched type untreated cells in transfusion medicine. Nevertheless, the quantities of enzymes used in these studies, even with present days most effective recombinant expression technology, renders ECO cells impractical mainly for economical reasons.
Claims of improved protocols for conversion of B cells using recombinant Glycine max α-galactosidase with a specific activity of approximately 200 U/mg have been reported using 5-10 units/ml with 16% hematocrit (U.S. Pat. Nos. 5,606,042; 5,633,130; 5,731,426; 6,184,017). The Glycine max α-galactosidase was thus used at 25-50 μg/ml, which represents a significant reduction in enzyme protein quantities required (50-200 fold) (Davis et al., Biochemistry and Molecular Biology International, 39(3): 471-485, 1996). This reduction is partly due to the higher specific activity of the Glycine max α-galactosidase (approximately 6 fold) as well as different methods used for conversion and evaluation. The 200 U/ml enzyme used in the study of Kruskall et al., (Transfusion, 40(11): 1290-8, 2000) was worked out for full unit (approximately 220 ml packed cells) conversions at 80-90% hematocrits and thoroughly analyzed by standard blood bank typing as well as by more sensitive cross-match analysis. Furthermore, the efficiency of conversion was evaluated by analysis of survival and induced immunity in patients receiving multiple transfusions of converted cells. The enzymatic conversions were done in test tubes in ml scale at 16% hematocrit, as described in U.S. Pat. Nos. 5,606,042 (and 5,633,130; 5,731,426; 6,184,017) with Glycine max (α-galactosidase, and the conversion efficiency not evaluated by cross-match analysis. Conversion of cells at 16% hematocrit required 10 U/ml, while conversions at 8% required 5 U/ml, indicating that converting at increased hematocrit requires more enzyme although higher cell concentrations were not tested. Thus, part of the reduction in enzyme protein quantities required compared to protocols reported by Kruskall et al., (Transfusion 2000; 40(11): 1290-8), is related to the concentration (hematocrit) of cells used in conversion, and this may represent more than 5-10 fold although direct comparison is not possible without experimentation. The U.S. Pat. No. 5,606,042 (and 5,633,130; 5,731,426; 6,184,017) further provides improvements in the conversion buffer using Na citrate and glycine at less acidic pH (preferably pH 5.8) and including additional protein in the form of BSA (bovine serum albumin) for stabilization. Interestingly, the conversion buffer developed for the Glycine max α-galactosidase was found not to be applicable to coffee bean α-galactosidase. Although, some improvement in the conversion of B cells may be provided by U.S. Pat. Nos. 5,606,042 (and 5,633,130; 5,731,426; 6,184,017), it is clear that at least more than 0.5 mg of enzyme is required per ml packed type B red cells using the disclosed protocol. It is likely that considerable more enzyme than this is required to obtain cells fully converted to O cells by the most sensitive typing procedures used in standard blood bank typing protocols. Furthermore, the protocol requires introduction of additional extraneous protein (BSA or human serum albumin) as well as exposing cells to acidic pH.
It is evident from the above that further improvements in conversion of B cells is required in order to make this a practical and commercially applicable technology. Necessary improvements include obtaining more efficient alpha-galactosidase enzymes, which allow conversion to take place preferable at neutral pH and without extraneous protein added.
Conversion of A Cells:
Levy and Animoff (J. Biol. Chem. 255: 1737-42, 1980) tested the ability of purified Clostridium perfringens α-N-acetylgalactosaminidase to convert A cells, and found reduction in antigen expression but considerable blood group A activity remained. Further studies of this enzyme have lead to purification to apparent homogeneity with a specific activity using the αGalNAc p-nitrophenyl substrate of 43.92 U/mg (Hsieh et al., IUBMB Life, 50(2): 91-7, 2000; PCT Application No. WO 99/23210). The purified enzyme had a neutral pH optimum with the αGalNAc p-nitrophenyl substrate, but no studies of the activity of this enzyme with oligosaccharides were presented. Some degradation of the A2 epitope with the purified enzyme in an ELISA assay was reported, but the enzyme have not been evaluated in enzyme conversion of A2 cells with appropriate blood typing.
Goldstein (Prog Clin Biol Res 165: 139-57, 1984; Transfus Med Rev 3(3): 206-12, 1989) was unsuccessful in converting A cells using chicken liver α-N-acetylgalactosaminidase. U.S. Pat. No. 4,609,627 entitled “Enzymatic Conversion of Certain Sub-Type A and AB Erythrocytes”, is directed to a process for converting Aint and A2 (including A2B erythrocytes) to erythrocytes of the H antigen type, as well as to compositions of type B erythrocytes which lack A antigens, which compositions, prior to treatment, contained both A and B antigens on the surface of said erythrocytes. The process for converting Aint and A2 erythrocytes to erythrocytes of the H antigen type, which is described in U.S. Pat. No. 4,609,627, includes the steps of equilibrating certain sub-type A or AB erythrocytes, contacting the equilibrated erythrocytes with purified chicken liver α-N-acetylgalactosaminidase enzyme for a period sufficient to convert the A antigen to the H antigen, removing the enzyme from the erythrocytes and re-equilibrating the erythrocytes. U.S. Pat. No. 6,228,631 entitled “Recombinant α-N-acetylgalactosaminidase enzyme and cDNA encoding said enzyme” provides a recombinant source for the chicken enzyme. The specific activities of purified and recombinant Pichia pastoris produced chicken liver α-N-acetylgalactosaminidase were reported to be approximately 51-56 U/mg using p-nitrophenyl αGalNAc as substrate (Zhu et al., Protein Expression and Purification 8: 456-62, 1996). The described conversion conditions for Aint and A2 cells in U.S. Pat. No. 4,609,627 included 180 U/ml cells (hematocrit not specified) at acidic pH 5.7, and treated cells did not agglutinate with unspecified anti-A reagent. This protocol requires more than 3 mg/ml enzyme protein and has not been reported to convert type A1 cells.
Hata et al. (Biochem Int. 28(1): 77-86, 1992) also reported conversion of A2 cells using chicken liver α-N-acetylgalactosaminidase at acidic pH. U.S. Pat. Nos. 5,606,042 (and 5,633,130; 5,731,426; 6,184,017) disclose similar results.
Falk et al. (Arch Biochem Biophys 290(2): 312-91991, 1991) demonstrated that an α-N-acetylgalactosaminidase purified from Ruminococcus torques strain IX-70 could destroy Dolichus biflorus agglutinability indicating that the A antigenic strength of A1 cells was reduced to the level of A2 cells.
Izumi et al. (Biochem Biophys Acta 1116: 72-74, 1992) tested purified Acremonium sp. α-N-acetylgalactosaminidase on type A1 cells. Although some reduction in agglutination titer was observed using 7,000 U/ml (140 U/20 μl) 4% hematocrit, conversion was not complete.
Human α-N-acetylgalactosaminidase enzyme has been isolated, cloned and expressed (Tsuji et al., Biochem. Biophys. Res. Commun. 163: 1498-1504, 1989, Wang et al., Human α-N-acetylgalactosaminidase-molecular cloning, nucleotide sequence, and expression of a full-length cDNA. Homology with human alpha-galactosidase A suggests evolution from a common ancestral gene. J Biol. Chem. 265: 21859-66, 1990) (U.S. Pat. No. 5,491,075). The pH optimum of human α-N-acetylgalactosaminidase is 3.5 (Dean K J, Sweeley C C. Studies on human liver alpha-galactosidases. II. Purification and enzymatic properties of alpha-galactosidase B ( alpha-N-acetylgalactosaminidase). J Biol. Chem. 254: 10001-5, 1979), similar to that of the human α-galactosidase (Dean K J, Sweeley C C. Studies on human liver alpha-galactosidases. I. Purification of alpha-galactosidase A and its enzymatic properties with glycolipid and oligosaccharide substrates. J Biol. Chem. 254: 9994-10000, 1979).
It is evident from the above that enzymatic conversion of type A cells, and particularly subgroup A1 cells constituting up to 80% of group A, has not been accomplished to date. Therefore, there exists a need in the prior art to identify appropriate enzymes capable of converting group A cells by removing all immunoreactive A antigens. Furthermore, there exists a need to develop appropriate conversion conditions preferably at neutral pH and without requirement of additional extraneous proteins.
Screening Assays:
Previous methods for searching, identification and characterization of exo-glycosidases have generally relied on the use of simple monosaccharide derivatives as substrates to identify saccharide and potential linkage specificity. Derivatized monosaccharide, or rarely oligosaccharide, substrates include without limitations p-nitrophenyl (pNP), benzyl (Bz), 4-methyl-umbrelliferyl (Umb), and 7-amino-4-methyl-coumarin (AMC). The use of such substrates provides easy, fast, and inexpensive tools to identify glycosidase activities, and makes large scale screening of diverse sources of enzymes practically applicable. However, the kinetic properties and fine substrate specificities of glycosidase enzymes may not necessarily be reflected in assays with such simple structures. It is also possible that novel enzymes with high degree of specificity and/or selective efficiency for complex oligosaccharide and unique glycoconjugate structures exists, but that these may have been overlooked and remain unrecognized due to methods of analysis. Thus, in order to identify and select the optimal exo-glycosidase for a particular complex oligosaccharide or glycoconjugate structure it may be preferable to use such complex structures in assays used for screening-sources of enzymes. Furthermore, assays used for screening may include selection for preferable kinetic properties such as pH requirement and performance on substrates, e.g., attached to the membrane of cells.
In the prior art, all α-galactosidases (EC 3.2.1.22) and α-N-acetylgalactosaminidases (EC 3.2.1.49) used for destroying B and A antigens of blood cells have been identified and characterized using primarily p-nitrophenyl monosaccharide derivatives. Interestingly, all α-galactosidase and α-N-acetylgalactosaminidase enzymes used in past studies to attempt removal of A and B antigens on cells are evolutionary homologous as evidenced by significant DNA and amino acid sequence similarities. Thus, the human α-galactosidase and α-N-acetylgalactosaminidase are close homologues (Wang et al., J Biol Chem, 265: 21859-66, 1990), and other enzymes previously used in blood cell conversion including the chicken liver α-N-acetylgalactosaminidase, fungal acremonium α-N-acetylgalactosaminidase, and bacterial α-galactosidases all exhibit significant sequence similarities. Primary structures of bacterial α-N-acetylgalactosaminidases have not been reported in the scientific literature. Because these glycosidases share sequence similarity it may be anticipated that the enzymes have related kinetic properties. Sequence analysis of all known O-glycoside hydrolases have been grouped in 85 distinct families based on sequence analysis, and the above mentioned α-galactosidases and α-N-acetylgalactosaminidases are grouped in families 27 and 36 (see, e.g., the webpage entitled “CAZy—Carbohydrate-Active Enzymes (Family GH32)” and located at afmb. cnrs-mrs.fr.about.cazy/CAZY/GH_32.html. These enzymes are characterized by having a retaining mechanism of catalysis and use aspartic acid as the catalytic nucleophile (Henrissat, Biochem Soc Trans, 26(2): 153-6, 1998; Rye & Withers, Curr Opin Chem Biol, 4(5): 573-80, 2000).
Therefore, there exists in the art a need to identify new α-galactosidase and α-N-acetylgalactosaminidase activities and corresponding enzyme proteins. If such enzymes exist, it is likely that they would not classify within families 27 and 36 because they would be selected to have significantly different kinetic properties.